Gene therapy for the treatment of HIV-1 infection and AIDS represents an attractive addition to conventional pharmacologic therapies due to the limited success of even highly active anti- retroviral therapies (HAART) and because genetic alteration of the host cell could potentially confer permanent suppression of viral replication after infection. Pre-clinical evaluation of new anti-HIV-1 gene therapies in an animal model is required to address many of current limitations of gene therapy including the duration of in vivo survival of transduced cells, loss of detectable anti-viral transgene expression and rapid clearing of transduced cells due to immune responses against the marker gene. Chimeric primate lentiviruses (SHIVs) composed of SIV and HIV genes have proven useful in the analysis of the role of discrete HIV genes in viral pathogenesis in macaque monkeys. Non-pathogenic SHIVs establish chronic infection and pathogenic SHIVs cause high levels of viremia, rapid and profound CD4+ T cell depletion and AIDS. Thus, the SHIV-macaque model offers a unique and important experimental system, to evaluate the effects of anti-viral genes without anti-viral Rx, to control input virus, to evaluate for development of resistance and to analyze for immune responses against the anti-viral gene products. We have characterized a human anti-tat intracellular single-chain antibody, termed sFvhutat2 "intrabody" with potent anti-HIV-1/SHIV activity in vitro and we now propose to test this gene therapy strategy in the SHIV-macaque model. In this proposal, we will optimize conditions for CD4+-enriched macaque T cell activation, transduction (with MuLV vectors either empty (control) or encoding sFvhutat2), phenotypic selection (via human NGFR) and ex vivo expansion and will complete in vitro challenge experiments to choose an isogeneic pair of non-pathogenic and pathogenic SHIVs that can be used for in vivo gene therapy studies. Both a pre-SHIV infection model and post-SHIV infection model are proposed using both non-pathogenic and pathogenic SHIVs in both models. For both models, we infuse equal numbers of both empty vector transduced or sFvhutat2 transduced CD4+-enriched T cells into 4 macaques (gene therapy arm) and mock transduced CD4+-enriched T cells into 4 macaques (control arm). The primary goal of these studies is to determine if there is increased survival of sFvhutat2 verses vector transduced cells. The secondary goal of these studies is to determine if in vivo resistance to sFvhutat2 develops. These studies will establish a valuable new primate model for the testing of anti-HIV-1 gene therapies. In addition, the results from these studies will substantially advance our understanding of how to apply and improve this promising technology for the treatment of HIV-1 infection and AIDS.